Single-gene studies, metagenomic assemblies, and the genome sequences of a limited number of cultured isolates are not a sufficient basis on which to accurately model the responses of natural microbial networks or engineer the function of artificial communities. For example, gene catalogs and composite genomes assembled from metagenomic data do not presently distinguish between genes that are tightly coupled within the context of the same organism and genes that are coupled across different organisms. This is a critical limitation, because only gene products encoded by the same organism can freely come into contact with one another to form complexes, drive signaling pathways, or carry out multi-step enzymatic transformations of diffusible substrates at maximum efficiency. A systems-level predictive understanding of microbial physiology absolutely demands the interpretation of genes and pathways in a full genomic context. Furthermore, individual organisms (indeed single cells) encoding full genomes are the basic replicating unit of biology and an important unit of evolutionary selection, factors that cannot be ignored in understanding the development of microbial networks in larger populations as a function of time.
In practical terms for single microbial cell sequencing, this means a million-fold amplification of the DNA present at the time of cell selection is required. Such high fold-amplification from sub-nanogram samples (Dean, et al., 2002, Proceedings of the National Academy of Sciences 99: 5261) and individual bacteria (Raghunathan, et al., 2005, Applied and Environmental Microbiology 71: 3342-3347) with good representation of the genome were first achieved by the multiple displacement amplification (MDA) whole-genome amplification (WGA) chemistry, but produced material with undesirable characteristics such as uneven representation and dislocated sequences. Nonetheless, investigators shortly succeeded in assembling shotgun sequence reads from single WGA-amplified E. coli and Prochlorococcus (Zhang, et al., 2006, Nat Biotechnol 24: 680-686), TM7 (Marcy, et al., 2007, Proc Natl Acad Sci USA 104: 11889-11894), and sequencing multiple genes from E. coli (Marcy, et al., 2007, PLoS Genet 3: 1702-1708), single marine bacteria (Stepanauskas & Sieracki, 2007, Proc Natl Acad Sci USA 104: 9052-9057), and soil and cultivated archaea (Kvist, et al., 2007, Applied microbiology and biotechnology 74: 926-935). Since the development of MDA, many other WGA methods have been developed and successfully applied to single cells (Blainey 2013, Cai & Walsh et al, 2012).
MDA is the WGA method that has been most commonly applied in single-cell sequencing of microbes. MDA works by the extension of 6-mer 3′-protected random primers on the DNA template (Dean, et al., 2001, Genome Res 11: 1095-1099). In MDA, a polymerase with strong strand displacement activity such as phi29 DNA polymerase or Bst DNA polymerase creates and displaces overlapping synthesis products from the template as single-stranded DNA under isothermal conditions (Dean, et al., 2001, Genome Res 11: 1095-1099; Zhang, et al., 2001, Mol Diagn 6: 141-150; Aviel-Ronen, et al., 2006, BMC Genomics 7). The displaced single-stranded DNA is a substrate for further priming and synthesis (Dean, et al, 2001, Genome Res 11: 1095-1099; Zhang, et al., 2001, Mol Diagn 6: 141-150). Phi29 DNA polymerase is typically specified for MDA due to its high accuracy owing to 3′-5′ exonuclease-mediated proofreading and exceptionally strong processivity in strand displacement synthesis, which can exceed 10,000 nt (Mellado, et al., 1980, Virology 104: 84-96; Blanco & Salas, 1984, Proceedings of the National Academy of Sciences 81: 5325; Blanco, et al., 1989, Journal of Biological Chemistry 264: 8935-8940; Morin, et al., 2012, Proceedings of the National Academy of Sciences 109: 8115-8120). This property of the polymerase evens out amplification on shorter genomic distances to produce high molecular weight products with more uniform amplification across the template than purely PCR-based methods, which typically produce products shorter than 1000 nt and exhibit greater amplification bias (Dean, et al., 2002, Proceedings of the National Academy of Sciences 99: 5261). In late 2012, one vendor started marketing a MDA kit that is decontaminated with ultraviolet light treatment and includes a mutant enzyme claimed to improve amplification uniformity and chimera performance (Qiagen REPLI-g Single Cell).
Given the concentration of contaminating fragments present in commercial WGA reagents (varying from 5 to 50 fragments per reaction microliter in the enzyme alone), volume reduction by itself does not necessarily eliminate reagent contamination (Blainey & Quake, 2011, Nucleic Acids Res 39: e19). For example, even driving reaction volumes down to the low nanoliter range, a significant fraction of reactions are still expected to carry contaminates from the reagents. Thus it is necessary to either inactivate contaminates in the reagents or produce reagent sets that are free of contamination. Contaminates in commercial WGA kits have been successfully suppressed by UV exposure with acceptable post-treatment amplification performance (Zhang, et al., 2006, Nat Biotechnol 24: 680-686; Woyke, et al., 2011, Plos One 6: e26161). Alternatively, reagents for background-free SVGA can be produced in batch processes utilizing disposable plasticware produced from virgin materials (Blainey & Quake, 2011, Nucleic Acids Res 39: e19). Irrespective of the clean-up approach taken, a key capability for validating reagent lots and clean-up procedures is a rapid assay for WGA activity and contamination. To be useful, the contamination assay must be both quantitative and extremely sensitive, such that different lots or treatments can be evaluated comparatively. QPCR is insufficiently sensitive since only contaminant molecules with an intact sequence locus matching specific PCR primers can be detected. For example, a PCR assay for the small subunit ribosomal RNA gene misses thousands of contaminant fragments arising from bacterial genomic DNA for every fragment molecule detected. Alternatively, the digital WGA (dWGA, eg digital MDA or dMDA) assay can be used for quantitation down to a few attograms of degraded genomic DNA per microliter (Blainey & Quake, 2011, Nucleic Acids Res 39: e19). dWGA is compatible with a variety of off-the-shelf platforms engineered for digital PCR (Baker, 2012, Nature methods 9: 541-544).
There is a need for improved methods for detecting contamination in reagents as well as improved methods for quantitating DNA.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.